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In Arabidopsis thaliana , biosynthesis of the essential thiol antioxidant, glutathione (GSH), is plastid-regulated, but many GSH functions, including heavy metal detoxification and plant defense activation, depend on cytosolic GSH. This finding suggests that plastid and cytosol thiol pools are closely integrated and we show that in Arabidopsis this integration requires a family of three plastid thiol transporters homologous to the Plasmodium falciparum chloroquine-resistance transporter, Pf CRT. Arabidopsis mutants lacking these transporters are heavy metal-sensitive, GSH-deficient, and hypersensitive to Phytophthora infection, confirming a direct requirement for correct GSH homeostasis in defense responses. Compartment-specific measurements of the glutathione redox potential using redox-sensitive GFP showed that knockout of the entire transporter family resulted in a more oxidized glutathione redox potential in the cytosol, but not in the plastids, indicating the GSH-deficient phenotype is restricted to the cytosolic compartment. Expression of the transporters in Xenopus oocytes confirmed that each can mediate GSH uptake. We conclude that these transporters play a significant role in regulating GSH levels and the redox potential of the cytosol.

Artemisinin-acridine hybrids were prepared and evaluated for their in vitro activity against tumour cell lines and a chloroquine sensitive strain of Plasmodium falciparum. They showed a 2-4-fold increase in activity against HL60, MDA-MB-231 and MCF-7 cells in comparison with dihydroartemisinin (DHA) and moderate antimalarial activity. Strong evidence that the compounds induce apoptosis in HL60 cells was obtained by flow cytometry, which indicated accumulation of cells in the G1 phase of the cell cycle. (C) 2009 Elsevier Ltd. All rights reserved.

On the basis of a mechanistic understanding of the toxicity of the 4-aminoquinoline amodiaquine (1b), three series of amodiaquine analogues have been prepared where the 4-aminophenol "metabolic alert" has been modified by replacement of the 4'-hydroxy group with a hydrogen, fluorine, or chlorine atom. Following antimalarial assessment and studies on mechanism of action, two candidates were selected for detailed ADME studies and in vitro and in vivo toxicological assessment. 4'-Fluoro-N-tert-butylamodiaquine (2k) was subsequently identified as a candidate for further development studies based on potent activity versus chloroquine-sensitive and resistant parasites, moderate to excellent oral bioavailability, low toxicity in in vitro studies, and an acceptable safety profile.

N-tert-Butyl isoquine (4) (GSK369796) is a 4-aminoquinoline drug candidate selected and developed as part of a public-private partnership between academics at Liverpool, MMV, and GSK pharmaceuticals. This molecule was rationally designed based on chemical, toxicological, pharmacokinetic, and pharmacodynamic considerations and was selected based on excellent activity against Plasmodium falciparum in vitro and rodent malaria parasites in vivo. The optimized chemistry delivered this novel synthetic quinoline in a two-step procedure from cheap and readily available starting materials. The molecule has a full industry standard preclinical development program allowing first into humans to proceed. Employing chloroquine (1) and amodiaquine (2) as comparator molecules in the preclinical plan, the first preclinical dossier of pharmacokinetic, toxicity, and safety pharmacology has also been established for the 4-aminoquinoline antimalarial class. These studies have revealed preclinical liabilities that have never translated into the human experience. This has resulted in the availability of critical information to other drug development teams interested in developing antimalarials within this class.

Acquired resistance to therapeutic agents is a major clinical concern in the prevention/treatment of malaria. The parasite has developed resistance to specific drugs through two mechanisms: mutations in target proteins such as dihydrofolate reductase and the bc1 complex for antifolates and nathoquinones, respectively, and alterations in predicted parasite transporter molecules such as P-glycoprotein homologue 1 (Pgh1) and Plasmodium falciparum CRT (PfCRT). Alterations in the expression of Pgh1 have been associated with modified susceptibility to a range of unrelated drugs. The molecular mechanism(s) that is responsible for this phenotype is unknown. We have shown previously (A. M. Ndifor, R. E. Howells, P. G. Bray, J. L. Ngu, and S. A. Ward, Antimicrob. Agents Chemother. 37:1318-1323, 2003) that the anticonvulsant phenobarbitone (PB) can induce reduced susceptibility to chloroquine (CQ) in P. falciparum , and in the current study, we provide the first evidence for a molecular mechanism underlying this phenomenon. We demonstrate that pretreatment with PB can elicit decreased susceptibility to CQ in both CQ-resistant and CQ-sensitive parasite lines and that this is associated with the increased expression of the drug transporter Pgh1 but not PfCRT. Furthermore, we have investigated the proximal promoter regions from both pfmdr1 and pfcrt and identified a number of putative binding sites for nuclear receptors with sequence similarities to regions known to be activated by PB in mammals. Whole-genome analysis has revealed a putative nuclear receptor gene, providing the first evidence that nuclear receptor-mediated responses to drug exposure may be a mechanism of gene regulation in P. falciparum .

The development of drug resistance to affordable drugs has contributed to a global increase in the number of deaths from malaria. This unacceptable situation has stimulated research for new drugs active against multidrug-resistant Plasmodium falciparum parasites. In this regard, we show here that deshydroxy-1-imino derivatives of acridine (i.e., dihydroacridinediones) are selective antimalarial drugs acting as potent (nanomolar K(i)) inhibitors of parasite mitochondrial bc(1) complex. Inhibition of the bc(1) complex led to a collapse of the mitochondrial membrane potential, resulting in cell death (IC(50) approximately 15 nM). The selectivity of one of the dihydroacridinediones against the parasite enzyme was some 5000-fold higher than for the human bc(1) complex, significantly higher ( approximately 200 fold) than that observed with atovaquone, a licensed bc(1)-specific antimalarial drug. Experiments performed with yeast manifesting mutations in the bc(1) complex reveal that binding is directed to the quinol oxidation site (Q(o)) of the bc(1) complex. This is supported by favorable binding energies for in silico docking of dihydroacridinediones to P. falciparum bc(1) Q(o). Dihydroacridinediones represent an entirely new class of bc(1) inhibitors and the potential of these compounds as novel antimalarial drugs is discussed.

Widespread parasitic resistance has led to an urgent need for the development and implementation of new drugs for the treatment of Plasmodium falciparum malaria. Artemisinin and its derivatives are becoming increasingly important, used preferably in combination with a second antimalarial agent to increase the efficacy and slow the development of resistance. However, cost, production and pharmacological issues associated with artemisinin derivatives and potential partner drugs are hindering the implementation of combination therapies. This article reviews the molecular basis of the action of, and resistance to, different antimalarials and examines the prospects for the next generation of drugs to combat this potentially lethal human pathogen.

In response to the emergence of parasite drug resistance to currently deployed antimalarials, the scientific community, in partnership with the pharmaceutical industry and public organizations, has fashioned an antimalarial drug development portfolio for the sustained development and registration of safe, effective and cheap antimalarial medicines. The management of this portfolio is being driven by MMV (Medicines for Malaria Venture), with a number of projects recently reaching the clinical end of this drug development pipeline.

In recent years, resistance to the antimalarial drug, chloroquine, has become widespread. It is, therefore, imperative to find compounds that could replace chloroquine or work synergistically with this drug to overcome chloroquine resistance. We have examined the interaction between chloroquine, a 4-aminoquinoline, and a number of 8-aminoquinolines, including primaquine, a drug that is widely used to treat Plasmodium vivax infections. We find that primaquine is a potent synergiser of the activity of chloroquine against chloroquine-resistant Plasmodium falciparum. Analysis of matched transfectants expressing mutant and wild-type alleles of the P. falciparum chloroquine resistance transporter (PfCRT) indicate that primaquine exerts its activity by blocking PfCRT, and thus enhancing chloroquine accumulation. Our data suggest that a novel formulation of two antimalarial drugs already licensed for use in humans could be used to treat chloroquine-resistant parasites.

Chloroquine resistance (CQR) in Plasmodium falciparum is associated with mutations in the digestive vacuole transmembrane protein PfCRT. However, the contribution of individual pfcrt mutations has not been clarified and other genes have been postulated to play a substantial role. Using allelic exchange, we show that removal of the single PfCRT amino-acid change K76T from resistant strains leads to wild-type levels of CQ susceptibility, increased binding of CQ to its target ferriprotoporphyrin IX in the digestive vacuole and loss of verapamil reversibility of CQ and quinine resistance. Our data also indicate that PfCRT mutations preceding residue 76 modulate the degree of verapamil reversibility in CQ-resistant lines. The K76T mutation accounts for earlier observations that CQR can be overcome by subtly altering the CQ side-chain length. Together, these findings establish PfCRT K76T as a critical component of CQR and suggest that CQ access to ferriprotoporphyrin IX is determined by drug–protein interactions involving this mutant residue.

Highly active antiretroviral therapy has substantially improved prognosis in human immunodeficiency virus (HIV). However, the integration of proviral DNA, development of viral resistance, and lack of permeability of drugs into sanctuary sites (e.g., brain and lymphocyte) are major limitations to current regimens. Previous studies have indicated that the antimalarial drug chloroquine (CQ) has antiviral efficacy and a synergism with HIV protease inhibitors. We have screened a panel of antimalarial compounds for activity against HIV-1 in vitro. A limited efficacy was observed for CQ, mefloquine (MQ), and mepacrine (MC). However, marked synergy was observed between MQ and saquinavir (SQV), but not CQ in U937 cells. Furthermore, enhancement of the antiviral activity of SQV and four other protease inhibitors (PIs) by MQ was observed in MT4 cells, indicating a class specific rather than a drug-specific phenomenon. We demonstrate that these observations are a result of inhibition of multiple drug efflux proteins by MQ and that MQ also displaces SQV from orosomucoid in vitro. Finally, coadministration of MQ and SQV in CD-1 mice dramatically altered the tissue distribution of SQV, resulting in a >3-fold and >2-fold increase in the tissue/blood ratio for brain and testis, respectively. This pharmacological enhancement of in vitro antiviral activity of PIs by MQ now warrants further examination in vivo.

New drugs are urgently needed to combat the growing problem of drug resistance in Plasmodium falciparum malaria. The infected erythrocyte is a multicompartmental system, and its transporters are of interest as drug targets in their own right and as potential routes for the delivery of antimalarial drugs. Choline is an important nutrient that penetrates infected erythrocyte membranes through the endogenous carrier and through parasite-induced permeability pathways, but nothing is known about its transport into the intracellular parasite. Here we present the first characterization of choline transport across the parasite membrane. Transport exhibits Michaelis-Menten kinetics with an apparent Km of 25.0 ± 3.5 μM for choline. The carrier is inhibitor-sensitive, temperature-dependent, and Na+-independent, and it is driven by the proton-motive force. Highly active bis-amidine and bis-quaternary ammonium compounds are also known to penetrate the host erythrocyte membrane through parasite-induced permeability pathways. Here, we demonstrate that the parasite choline transporter mediates the delivery of these compounds to the intracellular parasite. Thus, the induced permeability pathways in the host erythrocyte membrane and the parasite choline transporter described here form a cooperative transport system that shows great promise for the selective targeting of new agents for the chemotherapy of malaria. (Blood. 2004;104: 3372-3377)

Chloroquine resistance in Plasmodium falciparum is primarily conferred by mutations in pfcrt. Parasites resistant to chloroquine can display hypersensitivity to other antimalarials; however, the patterns of cross-resistance are complex, and the genetic basis has remained elusive. We show that stepwise selection for resistance to amantadine or halofantrine produced previously unknown pfcrt mutations (including S163R), which were associated with a loss of verapamil-reversible chloroquine resistance. This was accompanied by restoration of efficient chloroquine binding to hematin in these selected lines. This S163R mutation provides insight into a mechanism by which PfCRT could gate the transport of protonated chloroquine through the digestive vacuole membrane. Evidence for the presence of this mutation in a Southeast Asian isolate supports the argument for a broad role for PfCRT in determining levels of susceptibility to structurally diverse antimalarials.

Four series of C-10 non-acetal dimers were prepared from key trioxane alcohol 10beta-(2-hydroxyethyl)deoxoartemisinin (9b). All of the dimers prepared displayed potent low nanomolar antimalarial activity versus the K1 and HB3 strains of Plasmodium falciparum. The most potent compound assayed was phosphate dimer 14a, which was greater than 50 times more potent than the parent drug artemisinin and about 15 times more potent than the clinically used acetal artemether. In contrast to their potent activity versus malaria parasites, virtually all of the dimers expressed poor anticancer activity apart from the trioxane phosphate ester dimers 14a and 14b, which expressed nanomolar growth inhibitory (GI50) values versus a range of cancer cell lines in the NCI 60 human cell line screen. Further detailed studies on these dimers in vitro in HL60 cells demonstrate that both phosphate ester dimers (14a and 14b) are more potent than the anticancer agent doxorubicin. Interestingly, phosphate ester monomers 9c and 9d, antimalarially active in the low nanomolar region versus P. falciparum, are inactive as anticancer agents even at concentrations in the millimolar region. This observation emphasizes the importance of two trioxane units for high antiproliferative activity, and we propose that the nature of the linker in dimers of this type plays a crucial role in imparting potent anticancer activity.

A series of short chain chloroquine (CQ) derivatives have been synthesized in one step from readily available starting materials. The diethylamine function of CQ is replaced by shorter alkylamine groups (4-9) containing secondary or tertiary terminal nitrogens. Some of these derivatives are significantly more potent than CQ against a CQ resistant strain of Plasmodium falciparum in vitro. We conclude that the ability to accumulate at higher concentrations within the food vacuole of the parasite is an important parameter that dictates their potency against CQ sensitive and the chloroquine resistant K1 P. falciparum.

The maintenance of acidic pH in the digestive vacuole of the malaria parasite is thought to be crucial to the digestion of host cell haemoglobin and the subsequent process of heme detoxification. It may also be important in the mode of action of chloroquine and in the mechanism of resistance to the drug. Obtaining a definitive measurement of digestive vacuole pH has been surprisingly difficult. Some of the techniques for the measurement of pH in acid vesicles are outlined here along with some key aspects that are specific to malaria parasites. The use of acridine orange and dextran-tagged dyes as probes for the measurement of digestive vacuole pH has proved problematic, yet some surprising findings have emerged from work with these compounds.

The potent antimalarial activity of chloroquine against chloroquine-sensitive strains can be attributed, in part, to its high accumulation in the acidic environment of the heme-rich parasite food vacuole. A key component of this intraparasitic chloroquine accumulation mechanism is a weak base "ion-trapping" effect whereupon the basic drug is concentrated in the acidic food vacuole in its membrane-impermeable diprotonated form. By the incorporation of amino functionality into target artemisinin analogues, we hoped to prepare a new series of analogues that, by virtue of increased accumulation into the ferrous-rich vacuole, would display enhanced antimalarial potency. The initial part of the project focused on the preparation of piperazine-linked analogues (series 1 (7-16)). Antimalarial evaluation of these derivatives demonstrated potent activity versus both chloroquine-sensitive and chloroquine-resistant parasites. On the basis of these observations, we then set about preparing a series of C-10 carba-linked amino derivatives. Optimization of the key synthetic step using a newly developed coupling protocol provided a key intermediate, allyldeoxoartemisinin (17) in 90% yield. Further elaboration, in three steps, provided nine target C-10 carba analogues (series 2 (21-29)) in good overall yields. Antimalarial assessment demonstrated that these compounds were 4-fold more potent than artemisinin and about twice as active as artemether in vitro versus chloroquine-resistant parasites. On the basis of the products obtained from biomimetic Fe(II) degradation of the C-10 carba analogue (23), we propose that these analogues may have a mode of action subtly different from that of the parent drug artemisinin (series 1 (7-16)) and other C-10 ether derivatives such as artemether. Preliminary in vivo testing by the WHO demonstrated that four of these compounds are active orally at doses of less than 10 mg/kg. Since these analogues are available as water-soluble salts and cannot form dihydroartemisinin by P450-catalyzed oxidation, they represent useful leads that might prove to be superior to the currently used derivatives, artemether and artesunate.

Strong support for the NHE hypothesis is the use of the progeny from the genetic cross produced by Wellems et al.10xWellems, T.E. et al. Nature. 1990; 345: 253–255Crossref | PubMed | Scopus (354)See all References10 Lanzer's group demonstrated that saturable CQ uptake was reduced and cytosolic pH increased in the resistant progeny. Both of these observations can be explained by alternative mechanisms (see above). The main phenotypic characteristics that define CQ sensitivity and resistance, in both the genetic cross and in unrelated isolates, is the ability of verapamil to enhance CQ's accumulation and activity8xBray, P.G. et al. Mol. Pharmacol. 1998; 54: 170–179PubMedSee all References, 10xWellems, T.E. et al. Nature. 1990; 345: 253–255Crossref | PubMed | Scopus (354)See all References. Alignment of these observations with Lanzer's proposal dictates that verapamil, a known inhibitor of drug transporters and ion channels, must somehow restore the ability of the NHE of CQR parasites (which apparently is already working at maximal capacity) to be stimulated by drugs. This contrasts with recent experimental data demonstrating that the verapamil effect is independent of NHE activity in that the ability of verapamil to raise CQ accumulation in CQR parasites is retained in sodium-free medium8xBray, P.G. et al. Mol. Pharmacol. 1998; 54: 170–179PubMedSee all References8. The verapamil effect is perfectly linked with the CQ-resistant phenotype in a genetic cross10xWellems, T.E. et al. Nature. 1990; 345: 253–255Crossref | PubMed | Scopus (354)See all References10. Hence, if mutations of the parasite NHE are involved in CQ resistance, resistant parasites would have to evolve an independent mechanism for verapamil-stimulated drug uptake, simultaneously. This unlikely scenario suggests that mutations of the NHE are not responsible for the CQ-resistant phenotype. This reasoning is supported experimentally by the finding that, in sodium-free medium, the steady-state accumulation of CQ into CQS parasites is fourfold higher than that of CQR parasites8xBray, P.G. et al. Mol. Pharmacol. 1998; 54: 170–179PubMedSee all References8.In a further attempt to validate the NHE paradigm, Lanzer and his colleagues contended19xSanchez, C.P., Horrocks, P., and Lanzer, M. Cell. 1998; 92: 601–602Abstract | Full Text | Full Text PDF | PubMedSee all References19 that NHE is homologous to the recently identified cg2 parasite protein20xSu, X. et al. Cell. 1997; 91: 593–603Abstract | Full Text | Full Text PDF | PubMedSee all References20. Mutations in cg2 were shown to be associated with CQ resistance phenotypes. However, a deeper and more critical scrutiny failed to confirm any homology between the two proteins21xWellems, T.E. et al. Cell. 1998; 94: 285–286Abstract | Full Text | Full Text PDF | PubMed | Scopus (21)See all References21.Lanzer's group have taken care to present an intriguing alternative hypothesis to explain CQ resistance, and most importantly they have re-focused attention on CQ resistance and stimulated many people's interest. We feel that this is the correct time to highlight some of the experimental and conceptual inconsistencies that emerge from these investigations and to position this hypothesis alongside alternative explanations. It is our opinion that the NHE hypothesis has significant shortcomings. We hope that this commentary, rather than merely criticizing their data, will be the stimulus for the robust testing of all of the scientific issues raised.

Here we provide definitive evidence that chloroquine (CQ) uptake in Plasmodium falciparum is determined by binding to ferriprotoporphyrin IX (FPIX). Specific proteinase inhibitors that block the degradation of hemoglobin and stop the generation of FPIX also inhibit CQ uptake. Food vacuole enzymes can generate cell-free binding, using human hemoglobin as a substrate. This binding accounts for CQ uptake into intact cells and is subject to identical inhibitor specificity. Inhibition of CQ uptake by amiloride derivatives occurs because of inhibition of CQ–FPIX binding rather than inhibition of the Na+/H+ exchanger (NHE). Inhibition of parasite NHE using a sodium-free medium does not inhibit CQ uptake nor does it alter the ability of amilorides to inhibit uptake. CQ resistance is characterized by a reduced affinity of CQ–FPIX binding that is reversible by verapamil. Diverse compounds that are known to disrupt lysosomal pH can mimic the verapamil effect. These effects are seen in sodium-free medium and are not due to stimulation of the NHE. We propose that these compounds increase CQ accumulation and overcome CQ resistance by increasing the pH of lysosomes and endosomes, thereby causing an increased affinity of binding of CQ to FPIX.

We have investigated the contribution of drug accumulation and inhibition of heme polymerization to the in vitro activities of a series of antimalarial drugs. Only those compounds exhibiting structural relatedness to the quinolines inhibited heme polymerization. We could find no direct correlation between in vitro activity against chloroquine-susceptible or chloroquine-resistant isolates and either inhibition of heme polymerization or cellular drug accumulation for the drugs studied. However, in vitro activity against a chloroquine-susceptible isolate but not a chloroquine-resistant isolate showed a significant correlation with inhibition of heme polymerization when the activity was normalized for the extent of drug accumulation. The importance of these observations to the rational design of new quinoline-type drugs and the level of agreement of these conclusions with current views on quinoline drug action and resistance are discussed.

Aims: Chloroquine (CQ) kills Plasmodium falciparum by binding heme, preventing its detoxification to hemozoin in the digestive vacuole (DV) of the parasite. CQ resistance (CQR) is associated with mutations in the DV membrane protein P. falciparum chloroquine resistance transporter (PfCRT), mediating the leakage of CQ from the DV. However, additional factors are thought to contribute to the resistance phenotype. This study tested the hypothesis that there is a link between glutathione (GSH) and CQR. Results: Using isogenic parasite lines carrying wild-type or mutant pfcrt, we reveal lower levels of GSH in the mutant lines and enhanced sensitivity to the GSH synthesis inhibitor l-buthionine sulfoximine, without any alteration in cytosolic de novo GSH synthesis. Incubation with N-acetylcysteine resulted in increased GSH levels in all parasites, but only reduced susceptibility to CQ in PfCRT mutant-expressing lines. In support of a heme destruction mechanism involving GSH in CQR parasites, we also found lower hemozoin levels and reduced CQ binding in the CQR PfCRT-mutant lines. We further demonstrate via expression in Xenopus laevis oocytes that the mutant alleles of Pfcrt in CQR parasites selectively transport GSH. Innovation: We propose a mechanism whereby mutant pfcrt allows enhanced transport of GSH into the parasite's DV. The elevated levels of GSH in the DV reduce the level of free heme available for CQ binding, which mediates the lower susceptibility to CQ in the PfCRT mutant parasites. Conclusion: PfCRT has a dual role in CQR, facilitating both efflux of harmful CQ from the DV and influx of beneficial GSH into the DV. Antioxid. Redox Signal. 19, 683–695.

Four different series of triazole diamidines have been prepared by the Pinner method from the corresponding triazole dinitriles. Copper-catalyzed "click chemistry" was used for the synthesis of 1,4- and 4,5-substituted triazoles, aryl magnesium acetylide reagents for the 1,5-substituted triazoles, with a thermal dipolar addition reaction employed for the 2,4-substituted triazoles. In vitro antimalarial activity against two different PfCRT-modified parasite lines (Science 2002, 298, 210-213) of Plasmodium falciparum and inhibition of hemozoin formation were determined for each compound. Several diamidines with potent nanomolar antimalarial activities were identified, and selected molecules were resynthesized as their diamidoxime triazole prodrugs. One of these prodrugs, OB216, proved to be highly potent in vivo with an ED50 value of 5 mg kg(-1) (po) and an observed 100 % cure rate (CD100) of just 10 mg kg(-1) by oral (po) administration in mice infected with P. vinckei.

Background Plasmodium falciparum chloroquine resistance (CQR) transporter protein (PfCRT) is known to be the important key of CQR. Recent studies have definitively demonstrated a link between mutations in the gene pfcrt and resistance to chloroquine in P. falciparum. Although these mutations are predictive of chloroquine resistance, they are not quantitatively predictive of the degree of resistance. Methods In this study, a total of 95 recently adapted P. falciparum isolates from Thailand were included in the analysis. Parasites were characterized for their drug susceptibility phenotypes and genotypes with respect to pfcrt. From the original 95 isolates, 20 were selected for complete pfcrt sequence analysis. Results Almost all of the parasites characterized carried the previously reported mutations K76T, A220S, Q271E, N326S, I356T and R371I. On complete sequencing, isolates were identified with novel mutations at K76A and E198K. There was a suggestion that parasites carrying E198K were less resistant than those that did not. In addition, pfcrt and pfmdr1 gene expression were investigated by real-time PCR. No relationship between the expression level of either of these genes and response to drug was observed. Conclusion Data from the present study suggest that other genes must contribute to the degree of resistance once the resistance phenotype is established through mutations in pfcrt.

Cepharanthin is a proprietary extract of Stephania cepharantha , widely used in Japan for the treatment of inflammatory diseases. Cephranthin, its component alkaloids, and the standard resistance modulator verapamil were tested against Plasmodium falciparum for capacity to modulate sensitivity to chloroquine. Cepharanthin enhanced the activity of chloroquine against resistant clones by a factor of 15 at a concentration of only 200 nM (1.2 ng/ml). It is 50 times more potent than verapamil and 3 times more potent than the sum of its individual alkaloids. Combinations of component alkaloids acted synergistically to sensitize the parasite to chloroquine, possibly explaining the enhanced potency of Cepharanthin. Cepharanthin differed from verapamil in that it further sensitized clones that are considered to be fully susceptible, improving the baseline activity of chloroquine. Potent sensitization of parasites to chloroquine in vitro coupled with low toxicity suggests that coadministration of Cepharanthin might extend the clinical utility of chloroquine.

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The Plasmodium falciparum dihydrofolate reductase (PfDHFR) enzyme is the target of pyrimethamine, a component of the antimalarial pyrimethamine-sulfadoxine. Resistance to this drug is associated primarily with mutations in the Pf dhfr gene. The I164L mutant allele is of particular interest, because strains possessing this mutation are highly resistant to pyrimethamine and to chlorproguanil, a component of chlorproguanil-dapsone. A recent study from Malawi reported this mutation at a prevalence of 4.7% in parasites from human immunodeficiency virus-positive pregnant women by using a real-time PCR method. These observations have huge implications for the use of pyrimethamine-sulfadoxine, chlorproguanil-dapsone, and future antifolate-artemisinin combinations in Africa. It was imperative that this finding be rigorously tested. We identified a number of critical limitations in the original genotyping strategy. Using a refined and validated real-time PCR strategy, we report here that this mutation was absent in 158 isolates from Malawi and 42 isolates from Zambia collected between 2003 and 2005.

Malaria is a disease in desperate need of new chemotherapeutic approaches. Certain microtubule inhibitors, including vinblastine and taxol, have highly potent activity against malarial parasites and disrupt the normal microtubular structures of intra-erythrocytic parasites at relevant concentrations. While these inhibitors are useful tools, their potential as anti-malarial drugs is limited by their high toxicity to mammalian cells. In contrast, two classes of antimitotic herbicide, namely dinitroanilines (e.g. trifluralin and oryzalin) and phosphorothioamidates (e.g. amiprophosmethyl), exhibit moderate activity against the major human malarial parasite Plasmodium falciparum in culture but very low mammalian cytotoxicity. We examined the dynamics and kinetics of uptake and subcellular compartmentation of [14C]trifluralin in comparison with [3H]vinblastine. We wished to determine whether the relatively modest activity of trifluralin was the consequence of poor uptake into parasite cells. Trifluralin accumulated in parasite-infected erythrocytes to approximately 300 times the external concentration and vinblastine at up to approximately 110 times. Accumulation into uninfected erythrocytes was much lower. Uptake of trifluralin was rapid, non-saturable and readily reversed. It appears that the hydrophobic nature of trifluralin leads to accumulation largely in the membranes of the parasite, reducing the levels in the soluble fraction and limiting access to its microtubular target. By contrast, vinblastine accumulated predominantly in the soluble fraction and uptake was saturable and mostly irreversible, consistent with binding predominantly to tubulin. The results indicate that synthesis of more polar trifluralin derivatives may be a promising approach to designing microtubule inhibitors with more potent antimalarial activity.

Unsymmetrical dispiro- and spirotetraoxanes have been designed and synthesized via acid-catalyzed cyclocondensation of bis(hydroperoxides) with ketones. Incorporation of water-soluble and polar functionalities, via reductive amination and amide bond formation, produces several analogues with low nanomolar in vitro antimalarial activity. Several analogues display an unprecedented level of oral antimalarial activity for this class of endoperoxide drug.

Increase in drug detoxification and alteration of drug uptake and efflux of Plasmodium falciparum were investigated for their possible association with mefloquine (MQ) resistance in five different clones of P. falciparum from Thailand (T994b(3), K1CB(2), PR70CB(1), PR71CB(2) and TM(4)CB8-2.2.3). Fifty percent inhibitory concentration (IC(50)) values from these five clones varied between 30- and 50-fold. Regarding the detoxification mechanism, the ability of P. falciparum clones to biotransform MQ was shown in vitro by parasite microsomal protein prepared from parasite infected red blood cells protein (30mug), NADPH (1nM) and phosphate buffer pH 7.4, carried out at 37 degrees C with agitation. Radiolabelled unmetabolized MQ and possible metabolite(s) generated from the reaction was extracted into ethylacetate and separated by radiometric-HPLC after 1 h. All clones were capable of converting MQ into carboxymefloquine (CMQ), which is the main metabolite in human plasma. In addition, another unidentified metabolite eluted at 4.2 min on the chromatograph could be detected from the incubation reaction. This metabolite has never been detected in human liver microsomes before. There was no significant difference in the percentages of CMQ formed in the resistant (T994(b3), PR(70)CB(1), PR(71)CB(2)) and sensitive (TM(4)CB8-2.2.3, K1CB(2)) clones. Another possible mechanism, i.e., alteration in the accumulation of MQ in the parasites was investigated in vitro using [(14)C]MQ as a tracer. The time courses of [(14)C]MQ uptake and efflux were generally characterized by two phases. A trend of increased efflux of [(14)C]MQ was observed in the resistant compared with sensitive clones.

A leading bisthiazolium drug, T16, designed to mimic choline, was shown to exert potent antibabesial activity, with 50% inhibitory concentrations of 28 and 7 nM against Babesia divergens and B. canis , respectively. T16 accumulated inside Babesia -infected erythrocytes (cellular accumulation ratio, >60) by a saturable process with an apparent K m of 0.65 μM. Subcellular fractionation of Babesia parasites revealed the accumulation of T16 into a low-density fraction, while in malaria-infected erythrocytes a significant fraction of the drug was associated with heme malaria pigment. T16 exerts an early and specific inhibition of the de novo biosynthesis of phosphatidylcholine both in B. divergens- and Plasmodium falciparum -infected erythrocytes. Choline accumulation into isolated Babesia parasites was highly sensitive to inhibition by T16. These data are consistent with the hypothesis that bisthiazolium drugs target the de novo phosphatidylcholine biosynthesis of intraerythrocytic hematozoan parasites. In malaria parasites, which generate ferriprotoporphyrin IX during hemoglobin digestion, T16 binding to heme may enhance the accumulation and activity of the drug. The selectivity of accumulation and potent activity of this class of drug into parasite-infected erythrocytes offers unique advantages over more traditional antihematozoan drugs.

The aim of this study was to synthesise pure enantiomers of potent antimalarial 1,2,4-trioxanes, which are related to the natural antimalarial artemisinin, and then to assay each against a panel of Plasmodium falciparum strains. The working hypothesis was that if the artemisinin derivatives interact with a specific protein-target site, then there should be stereoselective differences in their activity. In five different P. falciparum isolates, however, the trioxane enantiomers (+)-7 a, (-)-7 a and (+)-7 b, (-)-7 b, showed the same level of in vitro antiparasitic activity.

We have adopted a proteomic strategy to investigate the actions of the two active components of the new antimalarial CoArtem, artemether and lumefantrine, following pharmacologically relevant drug exposure in the human malaria parasite Plasmodium falciparum. Both drugs induced profound alterations in the parasite's proteome. Moreover, the pattern of proteome alteration was specific for the drug used. The two drugs induced opposing effects on key glycolytic enzymes while exerting similar influence of the expression of stress response proteins. These initial results demonstrate the power of this approach in the study of pleiomorphic mechanisms of drug action.

The recent characterization of the choline carrier of the malaria parasite and its role in the selective delivery of novel antimalarial drugs has reignited interest in parasite transporters as a drug-delivery strategy. In this article, we discuss these findings in relation to the wider context of developing a sustainable antimalarial-drug-development portfolio.

Amodiaquine (AQ) (2) is a 4-aminoquinoline antimalarial that can cause adverse side effects including agranulocytosis and liver damage. The observed drug toxicity is believed to involve the formation of an electrophilic metabolite, amodiaquine quinoneimine (AQQI), which can bind to cellular macromolecules and initiate hypersensitivity reactions. We proposed that interchange of the 3' hydroxyl and the 4' Mannich side-chain function of amodiaquine would provide a new series of analogues that cannot form toxic quinoneimine metabolites via cytochrome P450-mediated metabolism. By a simple two-step procedure, 10 isomeric amodiaquine analogues were prepared and subsequently examined against the chloroquine resistant K1 and sensitive HB3 strains of Plasmodium falciparum in vitro. Several analogues displayed potent antimalarial activity against both strains. On the basis of the results of in vitro testing, isoquine (ISQ1 (3a)) (IC(50) = 6.01 nM +/- 8.0 versus K1 strain), the direct isomer of amodiaquine, was selected for in vivo antimalarial assessment. The potent in vitro antimalarial activity of isoquine was translated into excellent oral in vivo ED(50) activity of 1.6 and 3.7 mg/kg against the P. yoelii NS strain compared to 7.9 and 7.4 mg/kg for amodiaquine. Subsequent metabolism studies in the rat model demonstrated that isoquine does not undergo in vivo bioactivation, as evidenced by the complete lack of glutathione metabolites in bile. In sharp contrast to amodiaquine, isoquine (and Phase I metabolites) undergoes clearance by Phase II glucuronidation. On the basis of these promising initial studies, isoquine (ISQ1 (3a)) represents a new second generation lead worthy of further investigation as a cost-effective and potentially safer alternative to amodiaquine.

The burgeoning global problem of malaria is largely due to the emergence of parasite resistance to our limited armamentarium of antimalarial drugs. The recognition of this impending disaster at the international level and the engagement of the pharmaceutical industry promise a more optimistic future for antimalarial drug development. This is particularly exciting when considering the advances in our understanding of parasite biology, which are currently being fuelled by the malaria genome project. This article discusses recent developments in the area of antimalarial drug discovery and evaluation. New advances, based on traditional antimalarial drug classes including the quinolines, peroxides and antifolates ('back to the future'), are discussed, followed by a presentation of some novel targets ('young guns') that have been shown to be good candidates for chemotherapeutic attack.

Quaternary ammonium compounds have received recent attention due to their potent in vivo antimalarial activity based on their ability to inhibit de novo phosphatidylcholine synthesis. Here we show that in addition to this, heme binding significantly contributes to the antimalarial activity of these compounds. For the study, we used a recently synthesized bis-quaternary ammonium compound, T16 (1,12-dodecanemethylene bis[4-methyl-5-ethylthiazolium] diodide), which exhibits potent antimalarial activity (50% inhibitory concentration, ∼25 nM). Accumulation assays reveal that this compound is readily concentrated several hundredfold (cellular accumulation ratio, ∼500) into parasitized erythrocytes. Approximately 80% of the drug was shown to be distributed within the parasite, ∼50% of which was located in the parasite food vacuoles. T16 uptake was affected by anion substitution (permeation increasing in the order Cl − < Br − = NO 3 − < I − < SCN − ) and was sensitive to furosemide—properties similar to substrates of the induced new permeability pathway in infected erythrocytes. Scatchard plot analysis of in situ T16 binding revealed high-affinity and low-affinity binding sites. The high-affinity binding site K d was similar to that measured in vitro for T16 and ferriprotoporphyrin IX (FPIX) binding. Significantly, the capacity but not the K d of the high-affinity binding site was decreased by reducing the concentration of parasite FPIX. Decreasing the parasite FPIX pool also caused a marked antagonism of T16 antimalarial activity. In addition, T16 was also observed to associate with parasite hemozoin. Binding of T16 to FPIX in the digestive food vacuole is shown to be critical for drug accumulation and antimalarial activity. These data provide additional new mechanisms of antimalarial activity for this promising new class of antimalarial compounds.

Resistance to drugs can result from changes in drug transport, and this resistance can sometimes be overcome by a second drug that modifies the transport mechanisms of the cell. This strategy has been exploited to partly reverse resistance to chloroquine in Plasmodium falciparum . Studies with human tumor cells have shown that probenecid can reverse resistance to the antifolate methotrexate, but the potential for reversal of antifolate resistance has not been studied in P. falciparum. In the present study we tested the ability of probenecid to reverse antifolate resistance in P. falciparum in vitro. Probenecid, at concentrations that had no effect on parasite viability alone (50 μM), was shown to increase the sensitivity of a highly resistant parasite isolate to the antifolates pyrimethamine, sulfadoxine, chlorcycloguanil, and dapsone by seven-, five-, three-, and threefold, respectively. The equivalent effects against an antifolate-sensitive isolate were activity enhancements of approximately 3-, 6-, 1.2-, and 19-fold, respectively. Probenecid decreased the level of uptake of radiolabeled folic acid, suggesting a transport-based mechanism linked to folate salvage. When probenecid was tested with chloroquine, it chemosensitized the resistant isolate to chloroquine (i.e., enhanced the activity of chloroquine). This enhancement of activity was associated with increased levels of chloroquine accumulation. In conclusion, we have shown that probenecid can chemosensitize malaria parasites to antifolate compounds via a mechanism linked to reduced folate uptake. Notably, this effect is observed in both folate-sensitive and -resistant parasites. In contrast to the activities of antifolate compounds, the effect of probenecid on chloroquine sensitivity was selective for chloroquine-resistant parasites (patent P407595GB [W. P. Thompson & Co., Liverpool, United Kingdom] has been filed to protect this intellectual property).

The drug sensitivity characteristics and Plasmodium falciparum pfmdr1 status of five isolates of P. falciparum recently isolated from patients presenting for treatment from the Thailand/Myanmar border have been investigated. The aim of the study was to avoid the criticisms of some earlier studies by focusing on newly collected isolates from a specific geographic location. Three of the isolates studied exhibited clear resistance to chloroquine similar to that observed in the K1 Thai standard isolate obtained in the 1970s, and the other two isolates were of intermediate sensitivity to chloroquine with concentrations of drug that inhibit parasite growth by 50% of 50 and 43 nmol. The sensitivity of all isolates was enhanced by verapamil but we found no clear association between chloroquine sensitivity and gene copy number or intra-allelic variation of pfmdr1. In contrast, clear cross-resistance was seen between mefloquine and halofantrine, with the most sensitive isolates carrying the K1 mutation in pfmdr1.

The saturable uptake of chloroquine by parasites of Plasmodium falciparum has been attributed to specific carrier-mediated transport of chloroquine. It is suggested that chloroquine is transported in exchange for protons by the parasite membrane Na+/H+ exchanger [J Biol Chem 272:2652-2658 (1997)]. Once inside the parasite, it is proposed that chloroquine inhibits the polymerization of hematin, allowing this toxic hemoglobin metabolite to accumulate and kill the cell [Pharmacol Ther 57:203-235 (1993)]. To date, the contribution of these proposed mechanisms to the uptake and antimalarial activity of chloroquine has not been assessed. Using sodium-free medium, we demonstrate that chloroquine is not directly exchanged for protons by the plasmodial Na+/H+ exchanger. Furthermore, we show that saturable chloroquine uptake at equilibrium is due solely to the binding of chloroquine to hematin rather than active uptake: using Ro 40-4388, a potent and specific inhibitor of hemoglobin digestion and, by implication, hematin release, we demonstrate a concentration-dependent reduction in the number of chloroquine binding sites. An equal number of chloroquine binding sites are found in both resistant and susceptible clones, but the apparent affinity of chloroquine binding is found to correlate with drug activity (r2 = 0.93, p < 0.0001). This completely accounts for both the reduced drug accumulation and activity observed in resistant clones and the "reversal" of resistance produced by verapamil. The data presented here reconcile most of the available biochemical data from studies of the mode of action of chloroquine and the mechanism of chloroquine resistance. We show that the activity of chloroquine and amodiaquine is directly dependent on the saturable binding of the drugs to hematin and that the inhibition of hematin polymerization may be secondary to this binding. The chloroquine-resistance mechanism regulates the access of chloroquine to hematin. Our model is consistent with a resistance mechanism that acts specifically at the food vacuole to alter the binding of chloroquine to hematin rather than changing the active transport of chloroquine across the parasite plasma membrane.

The 4-aminoquinoline chloroquine (1) can be considered to be one of the most important synthetic chemotherapeutic agents in history. Since its discovery, chloroquine has proved to be a highly effective, safe, and well-tolerated drug for the treatment and prophylaxis of malaria. However, the emergence of chloroquine-resistant strains of the malarial parasite has underlined the requirement for a synthetic alternative to chloroquine. This review describes structure-activity relationships for the 4-aminoquinolines, along with views on the mechanism of action and parasite resistance. A description of drug metabolism and toxicity also is included, with a brief description of potential approaches to the design of new synthetic derivatives.

Plasmodium falciparum is the causative agent of the most deadly form of human malaria. Chemotherapy traditionally has been the main line of defence against this parasite, and chloroquine, the drug of choice, has been one of the most successful drugs ever developed. Unfortunately, the evolution and spread of resistance to chloroquine and other quinoline-containing drugs means that these compounds are now virtually useless in many endemic areas. Future prospects for the use of quinoline compounds improved considerably when it was demonstrated that chloroquine resistance could be circumvented in vitro by a number of structurally and functionally unrelated compounds such as verapamil and desipramine. The phenomenon of resistance reversal by compounds such as verapamil is also a key feature of drug resistance in mammalian cells, and this has raised the possibility that the underlying mechanisms of drug resistance of the two cell types could be similar. This hypothesis has prompted a large number of studies into the genetics and biochemistry of resistance to quinoline-containing drugs in P. falciparum. Both the genetic and the biochemical studies have raised issues of controversy and stimulated much debate. These issues are discussed in this review, in the context of a comparison with the genetics and biochemistry of multidrug resistance in mammalian cells.

Recent investigations into quinoline and phenanthrene methanol resistance in Plasmodium falciparum have described a linkage between amplification of the mdr homologue pfmdr1 and decreased susceptibility to mefloquine (MQ) and halofantrine (HF). We have examined the current theories on cross-resistance patterns and pfmdr1 gene expression by comparing the chloroquine (CQ) resistant isolate K1 with K1Hf, developed from the K1 isolate by intermittent exposure to halofantrine. Reduced halofantrine susceptibility in K1Hf was accompanied by reduced sensitivity to mefloquine and increased sensitivity to chloroquine. These sensitivity changes were reflected by changes in parasite drug accumulation. The loss of high level chloroquine resistance in K1Hf was associated with an inability of verapamil to enhance chloroquine sensitivity or accumulation. In contrast verapamil retained the chemosensitising potential against quinine in this isolate. The changes in phenotype were achieved without any amplification or increased expression of pfmdr1 or reversion of the Tyr86 mutation in the gene. Our data indicates that acquisition of halofantrine and mefloquine resistance and the loss of high level chloroquine resistance can be achieved without enhanced expression of the pfmdr1 gene product.

Amodiaquine is a 4-aminoquinoline antimalarial whose structure is similar to chloroquine. In contrast to the wealth of information available about chloroquine accumulation and its relationship to activity, little is known about the uptake characteristics of amodiaquine, a drug that is inherently more active against malaria parasites. In this study we have investigated the accumulation of amodiaquine in Plasmodium falciparum in vitro, in order to gain an insight into the mechanisms responsible for its superior activity over chloroquine. The driving force for parasite accumulation of the 4-aminoquinolines is proposed to be a transmembrane proton gradient maintained by a vacuolar ATPase. In the present study, amodiaquine accumulation was greatly reduced, at steady state, in the absence of glucose and at 0 degrees C indicating a clear energy dependence of uptake. Amodiaquine accumulation in Plasmodium falciparum was shown to be 2- to 3-fold greater than chloroquine accumulation. This observation probably accounts for amodiaquine's greater inherent activity but is surprising given that amodiaquine is a weaker base than chloroquine. With this in mind we present evidence for an intraparasitic binding component in the accumulation of the 4-aminoquinolines. Differences in binding affinity of this 'receptor' for amodiaquine and chloroquine may partially explain the greater accumulation and in vitro potency of amodiaquine compared to chloroquine.

The effect of artemether (AR) and quinine (QN) on parasite viability ex vivo was compared in children being treated for severe Plasmodium falciparum malaria. Parasitized blood taken at intervals during treatment was cultured in vitro, and parasite development was assessed microscopically. Parasite viability (defined as the proportion of circulating rings developing to early schizonts) was 56.8% in the AR group (n = 7) 6 hr after the start of treatment, compared with 93.3% for QN (n = 6; P = 0.015). Even after 24 hr of QN treatment, parasite viability was not significantly reduced in five patients. These ex vivo findings, which confirm previous observations of the stage-specific effects of these drugs against P. falciparum, suggest that AR may be superior to QN in the treatment of severe malaria.

The issue of chloroquine resistance in Plasmodium falciparum and cross-resistance patterns with other related chemotherapeutic agents has been the subject of intense interest for many years. Despite this level of investigation, the picture remains very unclear. Although it is accepted that chloroquine resistance is, at least in part, a function of reduced drug accumulation, the question of reduced drug uptake versus enhanced efflux is yet to be resolved at both the molecular and biochemical levels. Further, the absolute cross-resistance patterns of chloroquine-resistant isolates to closely related analogues is a matter for debate, although there appears to be a reciprocal arrangement between resistance to chloroquine and resistance to mefloquine, halofantrine and possibly quinine. Evidence is presented for the coexistence of two or more chloroquine-resistance mechanisms in isolates of P. falciparum, only one of which is verapamil sensitive. In addition, an analysis of cross-resistance patterns, as measured by the inoculum effect, is presented.

Aminoquinoline resistance correlates with lipid solubility at pH 7.2. Consequently, the in vivo dealkylation of amodiaquine, to the less lipid-soluble desethylamodiaquine, is a likely contributor to therapeutic failure in vivo. Therefore, 4-aminoquinoline drugs with lipid solubilities similar to that of amodiaquine, but which are not subject to side chain modification in vivo, should be superior antimalarial agents. In this study, we have identified amopyroquine and N-tertbutylamodiaquine as two such compounds. The values for the logarithms of the partition coefficients for amopyroquine and N-tertbutylamodiaquine are between those for amodiaquine and its dealkylated metabolite, desethylamodiaquine. Both amopyroquine and N-tertbutylamodiaquine possess levels of antimalarial activity greater than that of desethylamodiaquine and significantly reduced cross-resistance patterns; i.e., the former two compounds are not subject to the verapamil-sensitive resistance mechanism. Simple in vitro markers of direct toxicity and potential reactive metabolite formation suggest that these two compounds are no more toxic than amodiaquine and desethylamodiaquine.